From determining the differences between a healthy and diseased tissue, or one that has received treatment versus another or none at all, to understanding developmental signaling pathways, it’s hard to overestimate the importance of gene expression data in health and life sciences studies. Both in and of itself as a measure of RNA transcription, as well as a surrogate for protein expression, how much RNA of a given species is found, under which circumstances, and at which timepoint, can be a critical parameter.
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In this podcast, Erin Piazza, Senior Bioinformatics Scientist at NanoString, and Steven Ross, Vice President of IT at NanoString, describe how to effectively utilize gene expression data, as well as considerations for selecting the best profiling platform for your research.
Yet quantitating the relative numbers of transcripts present in sample can be a tedious procedure, fraught with human error as well as imprecision and biases built into the assays themselves. Most popular approaches, such as quantitative reverse transcription PCR (qRT-PCR) and RNA sequencing (RNA-Seq), and even the various microarray techniques, have technically challenging workflows that are highly reliant on enzymatic reactions and their associated biases.
This article looks at how an in-solution hybridization platform uses optical counting for enzyme-free digital gene expression investigations. The result is a user-friendly workflow with minimal hands-on time and quick turn-around time, yielding robust, easy-to-analyze data in a secure environment.
Rather than reverse-transcribing RNA and amplifying the resultant cDNA for PCR or sequencing, the NanoString nCounter© platform relies on optical barcodes to label the individual RNA molecules. Two contiguous DNA oligo probes—a capture probe containing biotin, and a reporter probe with a unique fluorescent barcode, each with a 50-base pair complementarity to the target molecule—are hybridized in solution to the target. These four pipetting steps per sample require only a few minutes of hands-on time, and are the only manual steps involved in the assay. Following an overnight incubation, the hybridization complexes are moved onto the automated platform where they are adhered to a streptavidin-coated slide and imaged, yielding reproducible results within just a few hours, for a total time-to-results of less than a day.
The barcodes, made up of six fluorophores, can be multiplexed to discriminate up to 800 unique molecular targets in each sample. Because each count of a barcode is a count of the RNA molecule to which it is hybridized, the raw digital barcode count is thus literally a raw count of labeled targets in the sample. Integrated analysis software allows the acquired data to be customized and formatted for presentation and publication. More advanced analyses can be performed using a free wizard-based add-on, while a free cloud-based platform offers guided modules for such tasks as cell type profiling, normalization, and quality control, enabling even users without bioinformatics or programming skills to analyze, interpret, and illustrate their findings.
nCounter has advantages typical of a digital gene expression platform, including wide dynamic range and high precision and reproducibility, and alleviating the need for technical replicates.
The platform requires only a 100-base pair region of RNA for hybridization. This helps it to be robust enough to handle difficult sample types including fragmented and degraded RNA found in formalin fixed paraffin embedded (FFPE)-preserved blocks routinely used to preserve clinical samples, as well as biofluids and cell lysates.
Researchers can take advantage of an extensive selection of off-the-shelf gene expression panels, targeted to a variety of pathways and research areas including oncology/IO, immunology, infectious disease, neuroscience, cardiovascular disease, and cell and gene therapy. Users can also customize their research by adding up to 55 user-defined targets to an existing panel.
High-throughput applications such as screening projects, for example, can benefit from nCounter’s PlexSet reagents. These allow for multiplexing of up to 96 pre-selected or custom probes across 96 samples, generating 9216 data points in a single run with only 30 minutes of hands-on time.
From defending against prying eyes and malicious hackers, to assuring that only the right users are accessing only the right files, protecting the integrity and security of data generated by a platform used for biomedical research is of utmost importance. The nCounter Pro Analysis system has all of the data integrity features expected from a modern system, including controlled access, hard drive and data encryption, and easy but secure data transfer and networking.
Data—including experimental parameters as well as results—are securely stored and auditable, with reports retrievable as part of a 21 CFR part 11 validated environment.
There are many ways to evaluate gene expression, each with its benefits and shortcomings. The NanoString nCounter offers an attractive balance of throughput, reliability, robustness, ease-of-use and -analysis, that makes it a formidable competitor in this space.